Amplification utilizing DNA primers and a DNA polymerase is a well known technique for detecting nucleic acid target sequences. Methods for exponential amplification include the polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and rolling-circle amplification (RCA). Among numerous DNA polymerases commonly used are Thermus aquaticus DNA polymerase and reverse transcriptase. The design of linear DNA oligonucleotide amplification primers is generally accomplished with the acid of a computer program designed for that purpose. Among the available programs that can be utilized are PRIDE (Haas et al. 1998), OLIGO (Rychlik et al. 1989), OSP (Hilber et al. 1991), Primo (Li et al. 1997) and Primer Master (Proutski et al. 1996).
A common problem is known as "primer-dimers". Primer-dimers are false amplification products (amplicons) that are generated because two primers hybridize to each other with overhangs, thereby providing binding sites for a polymerase and initiating DNA synthesis. Primer-dimers compete with the intended amplification and generally reduce the reliability and sensitivity of an assay. Another common problem is mis-priming of a sequence in a sample that is partially complementary to the primer. This also leads to false amplicons and reduces reliability and sensitivity.
One major application for target-amplification methods is in vitro diagnostics. In diagnosing pathological conditions by nucleic acid-based techniques, a common situation is that a unique nucleic acid sequence from a pathogen is a rare component of the total nucleic acid in a clinical sample. For example, the genomic DNA of the malarial parasite is a very small fraction of the total DNA that is extracted from a patient's blood. Amplification of rare pathogenic target sequences is an effective means for detection in some cases, because primers can be designed that successfully ignore the abundant human sequences sufficiently for diagnostic purposes. However, there are many situations in which a rare target sequence is very similar to an abundant sequence, differing in some cases by only a single nucleotide. For example, certain human cancers are characterized by an alteration at just one nucleotide position in a gene (Lengauer et al., 1998). To detect these cancers at an early stage, or to detect their remnants after surgical removal of a tumor, it is necessary to detect the presence of a rare sequence that differs from an abundant sequence by only a single nucleotide. When a sequence that indicates the presence of cancer is rare, the difficulty of detecting that sequence is sometimes referred to as the "minimal residual disease problem." A similar problem arises when the emergence of a drug-resistant bacterium or virus needs to be detected as early as possible when a patient is being treated with a drug, because a number of drug-resistance genotypes are characterized by a single nucleotide substitution in a pathogenic sequence. For applications such as those described above, simple target amplification is not effective, because the primers cannot sufficiently distinguish between two sequences that differ from each other by only a single nucleotide substitution.
Two approaches have been used to address this problem. The first is to design one of the two oligonucleotide primers that are needed for amplification to bind to the target at a sequence that encompasses the site of the nucleotide substitution. If the primer is perfectly complementary to its intended target sequence, then a primer-target hybrid will form, leading to the generation of amplified copies of the target nucleic acid sequence. The hope is that if a nucleotide substitution is present, then the mismatched primer-target hybrid will not form, resulting in an inability to generate amplified copies of the nucleic acid sequence. However, this dichotomy does not work well in practice, and both the mutant and the wild-type templates result in amplification. The products of amplification of perfect and mismatched targets (the "amplicons") are indistinguishable from one another. Even if only mismatched target sequences are present in the sample, the primer will occasionally initiate DNA synthesis on the mismatched target sequence. Because the resulting product contains a perfect complement of the primer sequence, exponential amplification of this initial product occurs at a rapid rate. The second approach that is used to detect mutations in a target sequence is to utilize primers that bind outside the sequence that might contain a mutation, so that the sequence that contains the site of the mutation becomes a part of the resulting amplicons. Additional hybridization probes are then used to determine if the mutation is present within the amplicons. The proportion of amplicons containing a mutation is a measure of the relative amount or absolute amount of the mutation in the starting sample. Although this approach works well in many situations (Tyagi et al. 1996, Tyagi et al. 1998), it has a sensitivity limitation: if the mutant amplicons are less than a few percent of all the amplicons, they cannot be detected.
In order to detect mutants that are rare, that is, less abundant than the few percent of the wild-type sequence that is needed for detection by hybridization probes, an "amplification refractory mutation system" (ARMS) has been used (Newton et al., 1989; Wu et al., 1989). In this method, two amplification reactions are carried out in separate reaction tubes. The difference between the reactions is that one of the primers is slightly different in each tube. The difference between the primers is in the identity of the nucleotide at their 3' ends. The 3' nucleotide of the primer in one reaction tube is complementary to the wild-type nucleotide at the site of mutation, while the 3' nucleotide of the primer in the other reaction tube is complementary to the mutant nucleotide at the site of mutation. If the primer in the tube is perfectly complementary to its target sequence, including the nucleotide at the 3'-end of the primer, then the primer can be efficiently extended by incubation with DNA polymerase. However, if the binding of the primer in the tube to the target sequence creates a mismatched 3'-terminal nucleotide, then the primer cannot be efficiently extended by incubation with DNA polymerase. Amplification of the mismatched template is significantly delayed, i.e., the number of thermal cycles in a polymerase chain reaction (PCR) amplification that are required before the amplification product can be detected (or the amount of time it takes to generate a detectable quantity of amplification product in an isothermal amplification) is significantly greater when the 3' nucleotide of the primer is not complementary to the sequence present in the sample.
ARMS primers and conventional primers are both prone to generating false-positive signals, because they can initiate the exponential synthesis of unintended amplicons, even in absence of perfectly complementary target sequences. These "false amplicons" arise because the 3' end regions of the primers can bind to partially complementary sequences unrelated to the target that are present in samples. They also can arise from the binding of one primer molecule to another primer molecule, which results in the initiation of DNA synthesis (primer-dimers). In either case, the resulting extension products can be exponentially amplified in the normal manner, resulting in the synthesis of false amplicons. The generation of false amplicons not only makes it difficult to identify the intended amplicons, but also limits the sensitivity of assays, since false amplicons compete with the intended amplicons, and thereby reduce their abundance. For example, if a rare target sequence requires 38 cycles of PCR to be detectable above background, but a false amplicon in the reaction becomes detectable after 35 cycles, the rare target will not reach a detectable level.
The present invention markedly improves the specificity of oligonucleotide primers.
One aspect of the invention is an improvement in the sensitivity of assays that detect target nucleic acids that contain a single nucleotide substitution within a population of much more abundant wild-type nucleic acids, enabling detection at levels below a few percent.
Another aspect of the invention is a reduction in the formation of false amplification products, including primer-dimers.
Another aspect of the invention is that it enables the determination of the fraction of a nucleic acid population that is mutant and the fraction that is wild type, particularly when the fraction is very small or very large.
Another aspect of the invention is the decrease or elimination of the tendency of target amplification reactions to produce false amplicons.
Another aspect of the invention is to provide a means of labeling the amplification product with a fluorescent moiety so that the reactions can be monitored in real time without having to utilize probes or nonspecific intercalating reagents.